Embryonic stem cells (ESCs) are unspecialized cells that have the ability to self-renew, producing daughter cells with equivalent developmental potential, or to differentiate into more specialized cells. ESCs are derived from the inner cell mass of the pre-implantation embryo and are pluripotent, as they are able to differentiate in vivo into all cell types of the adult organism, but not into extraembryonic tissue. Control over cell fate decisions is accomplished through a variety of poorly defined molecular, genetic and epigenetic events.
Exogenous control of the pluripotent state can be achieved by a limited number of factors. When grown in fetal bovine serum (FBS)-containing medium and in the presence of murine embryonic fibroblast feeder cells or the cytokine leukemia inhibitory factor (LIF), mouse ESCs remain undifferentiated. LIF functions through the activation of gp130 signaling through binding to LIFRβ. LIFRβ then dimerizes with gp130 and transduces the signal through the JAK-STAT pathway and is thought to maintain the undifferentiated state through inhibition of mesoderm and endoderm formation. While STAT3 plays an important role in self-renewal of mouse ESCs, Stat3−/− embryos can undergo gastrulation, suggesting the existence of a STAT3-independent pathway for pluripotent stem cell self-renewal. Another factor, BMP4, provided by the serum, functions in the presence of LIF to maintain pluripotency by inducing phosphorylation and nuclear localization of Smad1, followed by up-regulation of Id proteins that block neural differentiation.
Three transcription factors are known to be critical in the establishment and/or maintenance of ESC pluripotency: Oct4, Nanog and Sox2. OCT4 (Pou5f1) has a highly conserved role in maintaining pluripotent cell populations and its expression level dictates ESC fate. SOX2 forms a complex with OCT4 and is necessary to co-operatively activate target genes in ESCs. These factors comprise one essential circuit regulating ESC pluripotency in which OCT4 regulates Sox2, and additionally, the OCT4-SOX2 complex activates Oct4 expression. Forced over-expression of Nanog maintains pluripotency and OCT4 levels in ESCs, even in the absence of LIF while it is itself regulated by OCT4 and SOX2. All three factors are down-regulated during differentiation induced by LIF withdrawal or retinoic acid (RA) induction. Genome-wide analysis of Oct4, Nanog, and Sox2 transcriptional targets illustrate that they regulate a plethora of genes implicated in numerous cellular pathways and functions. These genes along with c-myc and Klf4 play critical roles in the reprogramming of fibroblasts into induced pluripotent (iPS) cells. These same transcription factors are also implicated in tumour progression of cancers such as germ cell tumours, embryonal and breast carcinomas, and are currently being investigated as neoplastic markers.
Epigenetics refers to the acquisition of heritable traits that do not involve changes to the underlying genomic structure. Recent studies have revealed that epigenetic processes, such as DNA and histone methylation are also crucial determinants of cellular differentiation and help explain how the single genome of a stem cell can be actively modified to produce differentiated progeny with diverse cellular identities. The chromatin state or ‘epigenome’ of ESCs is largely void of DNA methylation but possesses histone modifications, in particular, methylation of histone H3 at lysine 4 (H3K4) and H3 at lysine 27 (H3K27). Di-methylation (2me-) at H3K4 is a transcriptionally active mark, whereas tri-methylation (3me-) of H3K27 is a repressive mark. This “bivalent” mark is believed to hold the chromatin in a transcriptionally ready state and upon specific stimulus the mark will be resolved as either activated or repressed, resulting in increased or decreased gene transcription.
The Polycomb group (PcG) proteins are regulators of the epigenetic state of the cell. These proteins exist in one of two main complexes, the maintenance complex Polycomb Repressive Complex 1 (PRC1) and the initiation complex Polycomb Repressive Complex 2 (PRC2). The core components of PRC2 are EED, EZH2 and SUZ12. Through the methyltransferase activity of the EZH2 protein, the PRC2 complex deposits the repressive mark of tri-methylation on lysine 27 of histone 3 (3me-H3K27). The PRC1 complex is thought to recognize the 3me-H3K27 histone modification and subsequently methylate the chromatin at that promoter to ensure stable, long-term silencing.
Ablation of PRC2 components results in embryonic lethality and defects in histone methylation and cell proliferation. Suz12-null ESCs are able to maintain an undifferentiated morphology but are unable to differentiate into mature cell types and maintain high levels of ESC markers even after the withdrawal of self-renewal signals. A recent report shows that Eed null ESCs express heightened levels of target differentiation genes but also maintain high levels of ESC markers and can be taken to high passage without losing their undifferentiated morphology. Ezh2, Eed, and Suz12 are downstream targets of the pRb/E2F pathway further indicating a role in proliferation and are up-regulated in cancers including lymphomas, prostate and breast cancers. Potential roles of PRC2 in tumorigenesis include suppression of cyclin-dependent kinase inhibitor p16ink4a expression and promoter hypermethylation.
Expression of PRC1 components, including Bmi1 and Mel18, is also deregulated in a number of tumour types. BMI1 possesses oncogenic properties when over-expressed and contributes to tumorigenesis by inhibiting expression of p16ink4a. In contrast, MEL18 appears to function as a tumour suppressor by repressing the expression of Bmi1 in breast cancer cells. Interestingly, BMI1 is required for maintenance of the hematopoietic and neural stem cell lineages and deletion of Bmi1 leads to pleiotropic defects and postnatal death by 20 weeks of age.
The highly conserved Pcl2 gene interacts with PRC2 by associating with EZH2 and plays a role in embryonic patterning in chick. The Pcl2 gene encodes a TUDOR domain and two plant homeodomain (PHD) type zinc fingers. The homeodomain PHD finger is found in nuclear proteins thought to be involved in chromatin-mediated transcriptional regulation. It is involved in protein-protein interaction and important for the assembly or activity of multicomponent complexes involved in transcriptional activation or repression. The TUDOR domain may bind to RNA or ssDNA or may control interactions with protein complexes.
In general, epigenetic alterations such as changes to DNA methylation and/or chromatin structure have been implicated in the pathogenesis and progression of neoplasia. Genome-wide DNA hypomethylation and regionalized promoter hypermethylation leads to genomic instability and repression of tumour-suppressor genes (TSGs), respectively, both hallmarks of cancers. The importance of epigenetic perturbations in neoplasia is highlighted by the fact that methylation and histone-modifying drugs have the capacity to inhibit malignancy in a number of cancer types. Importantly, tri-methylated H3K27 and di-methylated H3K9 are associated with gene promoters whose DNA is frequently hypermethylated and thus repressed in adult cancers.
The process of asymmetric stem cell division is highly regulated and perturbations in cell fate decisions can lead to a variety of disorders including developmental defects, degenerative disease and cancer. The identification of molecules with key roles in regulating ESC pluripotency is critical to provide an improved understanding of the molecular pathways responsible for maintenance of the stem cell phenotype. In addition, information regarding stem cell markers can be used to identify potential therapeutic targets.
It would be desirable, thus, to further understand the factors which control the pluripotent state.